In order to advance our understanding of the chemistry and biology of the greater family of manganese- requiring enzymes, we propose to explore structure-function relationships in human arginase I as well as the arginase-related metalloenzymes histone deacetylase 8 and polyamine deacetylase. Human arginase I contains a binuclear manganese cluster required for the hydrolysis of L-arginine to form L-ornithine and urea, and our studies indicate that catalysis proceeds through a mechanism in which both metal ions function to activate a metal-bridging hydroxide ion as the catalytic nucleophile. We have determined the structure of this enzyme to 1.29 resolution, and we will use this structure to guide the design of inhibitors and biosensors. Recent discoveries show that arginase is upregulated in various diseases such as atherosclerosis, asthma, and cancer, so our studies will expand the repertoire of chemical compounds that will potentially be useful for the treatment and diagnosis of human disease. Given the newly-discovered and unexpected structural relationship between the arginases and metal- dependent deacetylases, our structural and functional studies will illuminate important mechanistic parallels between these enzyme families. Intriguingly, the Zn2+ site of the deacetylase corresponds to the Mn2+B site of arginase, but the deacetylase does not contain a metal binding site corresponding to Mn2+A of arginase. Thus, the stoichiometry of metal binding has diverged in the evolution of the arginases and the deacetylases from a common metalloenzyme precursor. Intriguingly, the biologically preferred metal ion of human histone deacetylase-8 is believed to be Fe2+. Therefore, we will determine the structures of the Fe2+-substituted enzyme, its site-specific variants, and its substrate and inhibitor complexes. Since this enzyme is a validated drug target for cancer chemotherapy, it is important to thoroughly understand structure-function relationships in the form of the metalloenzyme that is found in vivo. Overall, the proposed research will provide a greater structural and functional understanding of metal ion specificity (Mn2+, Zn2+, Fe2+) and stoichiometry in the evolution of the arginases and the arginase-related deacetylases.